USPTO Patents Application 09993647

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(19) World Intellectual Property Organization

International Bureau

(43) International Publication Date
18 May 2007 (18.05.2007)

PCT

(10) International Publication Number

wo 2007/056011 A2

(51) International Patent Classification:
GOIN 33/574 (2006.01)

(21) International Application Number:

PCT/US2006/042660

(22) International Filing Date:

1 November 2006 (01.11.2006)

(25) Filing Language: English

(26) Publication Language: English

(30) Priority Data:

60/733,100 2 November 2005 (02.1 1.2005) US

(71) Applicant (for all designated States except US): BAYER
HEALTHCARE LLC [US/US] ; DIAGNOSTICS DIVI-
SION, 511 BENEDICT AVENUE, Tarrytown, NY 10591-
5097 (US).

(72) Inventors; and

(75) Inventors/Applicants (for US only): ELTING, James,

J. [US/US]; 5 HEATHERWOOD DRIVE, Madison,
CT 06443 (US). CARNEY, Walter, P. [US/US]; 14
BAY STATE ROAD, North Andover, MA 01845 (US).
HAMER, Peter, J. [US/US]; 25 DUDLEY STREET,
Reading, MA 01867 (US). BIGWOOD, Douglas
[US/US]; 72 KELSEY SPRINGS DRIVE, Madison, CT
06443 (US).

(74) Agent: STEIN, Kevin; BAYER HEALTHCARE LLC,
LAW & PATENTS DEPARTMENT, 511 BENEDICT AV-
ENUE, Tarrytown, NY 10591-5097 (US).

(81) Designated States (unless otherwise indicated, for every
kind of national protection available): AE, AG, AL, AM,
AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI,
GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS,
JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS,
LT, LU, LV, LY, MA, MD, MG, MK, MN, MW, MX, MY,
MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT, RO, RS,
RU, SC, SD, SE, SG, SK, SL, SM, SV, SY, TJ, TM, TN,
TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.

(84) Designated States (unless otherwise indicated, for every
kind of regional protection available): ARIPO (BW, GH,
GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
FR, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, NL, PL, PT,
RO, SE, SI, SK, TR), OAPI (BE, BJ, CF, CG, CI, CM, GA,
GN, GQ, GW, ML, MR, NE, SN, TD, TG).

Published:

— without international search report and to be republished
upon receipt of that report

[ Continued on next page ]

(54) Title: METHODS FOR PREDICTION AND PROGNOSIS OF CANCER, AND MONITORING CANCER THERAPY

I I Sorafenib

Placebo

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VEGF (pg/ml)

330

280-

230 -

180-

130 -

80

Baseline

Cycle 1,
Day 21

Cycle 3,
Day 1

(57) Abstract: The present invention relates to biomarkers and the use of biomarkers for the prediction and prognosis of cancer as
well as the use of biomarkers to monitor the efficacy of cancer treatment. Specifically, this invention relates to the use of VEGF as
a biomarker for multi-kinase inhibitors.

wo 2007/056011 A2 llllllllllllllllllllllllllllllllllllllllllllllll

For two -letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette.

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METHODS FOR PREDICTION AND PROGNOSIS OF CANCER,

AND MONITORING CANCER THERAPY

FIELD OF THE INVENTION

[001] The present invention relates to biomarkers and the use of biomarkers for the
prediction and prognosis of cancer as well as the use of bionnarkers to monitor the efficacy of
cancer treatment Specifically, this invention relates to the use of VEGF as a biomarker for
multi-kinase inhibitors.

BACKGROUND OF THE INVENTION

[0021 Vascular endothelial gro\Arth factor receptors (VEGFRs) and their ligands, vascular
endothelial growth factors (VEGFs), play critical roles in endothelial cell migration and
proliferation. The VEGFRA/EGF system includes three receptors (VEGFR-1 , VEGFR-2, and,
VEGFR-3) and four ligands (VEGF-A, B, C. D, and E and placental growth factor). VEGF-A
further consists of four isoforms, VEGF-121, VEGF-165, VEGF-185, and VEGF-204, derived
from alternative transcription of the VEGF-A gene. The receptors are plasma membrane-
spanning proteins with intracellular tyrosine kinase domains. As with other protein kinases,
activation of the VEGFRs is a key mechanism in regulating signals for endothelial cell
proliferation, and abnormalities of VEGFRA/EGF are thought to contribute to abnormal
angiogenesis in number of human diseases such as psoriosis and malignancy.

[003] In embryogenesis, the VEGFRA/EGF system is essential for the correct development
of the vascular system. In adults, VEGFR/VEGF is important in wound healing,
inflammation, and angiogenesis.

[004] A noninvasive assay for circulating VEGF levels in patients prior to drug treatment is
a potentially important adjunct to therapeutic decision making. Although assays of total
VEGF-A have been used in humans as a prognostic indicator of disease outcome, until the
instant disclosure, no correlation between levels of VEGF in patients prior to chemotherapy
and treatment outcome have been reported. Therefore, VEGF may serve as a valuable

*

prognostic indicator, and as a biomarker to monitor the efficacy of treatment with a multi-
kinase inhibitor.

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SUMMARY OF THE INVENTION

[005] The present invention relates to biomarkers and the use of biomarkers for the
prediction and prognosis of cancer as well as the use of bionaarkers to nnonitor the efficacy of
cancer treatment. Specifically, this invention relates to the use of VEGF as a biomarker for a
5 multi-kinase inhibitor (e.g., Sorafenib).

[006] In one ennbodiment, the present invention relates to the use of quantitative
imnnunoassays to measure levels of VEGF protein in human body fluids prior to treatment
with a multi-kinase inhibitor (e.g., Sorafenib). Said levels are particularly useful as an
indicator of the potential for cancer patients treated with a multi-kinase inhibitor (e.g.,
10 Sorafenib) to benefit from such therapy.

[007] Measurement of post-treatment levels of VEGF, as well as the change in VEGF
levels over the course of treatment, can be used clinically as a therapeutic aid for patient
therapy selection, to monitor the status of a preneoplastic/neoplastic disease in a patient,
and/or to monitor how a patient with a preneoplastic/neoplastic disease is responding to a
15 therapy. In one embodiment, the levels of VEGF may be used to aid in patient therapy
selection, and to make decisions about the optimal method for patient therapy.

[008] The levels of VEGF may be measured in patient samples such as, but not limited to,
blood, serum, plasma, urine, saliva, semen, breast exudate, cerebrospinal fluid, tears,
sputum, mucous, lymph, cytosols, ascites, pleural effusions, amniotic fluid, bladder washes,
20 and bronchioalveolar lavages.

[009] In another embodiment, the invention relates to the use of an immunoassay as a
method of selecting patients who are likely to benefit from multi-kinase inhibitor (e.g.,
Sorafenib) treatment by measuring pretreatment levels of VEGF in patient samples and
assessing probable outcome based on a nomogram of likely patient outcome versus VEGF
25 levels.

[010] A method of monitoring the status of a disease associated with an activated VEGF
pathway in a patient may be further prognostic for a disease, wherein the levels of total
VEGF protein in the patient's samples are indicative of a better or poorer treatment outcome
for the patient. The prognosis may be a clinical outcome selected from the group consisting
30 of response rate (RR), complete response (CR), partial response (PR), stable disease (SD),
clinical benefit [including complete response (CR), partial response (PR), and stable disease
(SD)], time to progression (TTP), progression free survival (PFS), and overall survival (OS).

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[011] These methods may be in standard formats, for example, an immunoassay in the
form of a sandwich immunoassay, such as a sandwich enzyme-linked immunosorbent assay
(ELISA) or an equivalent assay. These immunoassays may use monoclonal antibodies,
such as anti-VEGF monoclonal antibodies. Furthermore, the monoclonal antibody may be
5 biotinylated.

[012] Another embodiment of the invention relates to a quantitative immunoassay to
measure serial changes in the levels of total VEGF protein in patient samples, as a method
of therapy selection for a patient with a disease, for example, a preneoplastic/neoplastic
disease.

10 [013] As an example, one such method of therapy selection may comprises the steps of:

(a) immunologically detecting and quantifying the level of total VEGF protein in a sample
from a control population;

(b) immunologically detecting and quantifying the level of total VEGF protein in samples
taken from a patient over time; and

15 (c) determining whether to use conventional therapy and/or multi-kinase inhibitor (e.g.,

Sorafenib) therapy to treat the patient based the level of VEGF protein in the patient's
samples.

[014] For example, if the level of VEGF protein in a patient's sample is found to be above
70 pg/ml, the conclusion could be drawn that the patient has a VEGF driven disease, and
20 the decision may be made to use multi-kinase inhibitor (e.g., Sorafenib) therapy to treat the
patient, either alone or in conjunction with one or more other therapies.

[015] A VEGF pathway-directed therapy may be multi-kinase inhibitors, tyrosine kinase
inhibitors, bis-aryl ureas, antisense inhibitors of VEGFR-2, or monoclonal antibody therapies,
or the like. For example, a VEGF pathway-directed therapy may be the bis-aryl urea
25 Sorafenib, which is an angiogenesis inhibitor as well as a tyrosine kinase inhibitor, or the
tyrosine kinase inhibitor, STI571 (also known as imatinib mesylate or Gleevec®).

[016] Another embodiment of the invention relates to the use of quantitative immunoassays
to detect changes in VEGF levels in combination with the levels of one or more other
protein(s). Such additional protein(s) may include, for example, inhibitors (e.g., tissue-
30 inhibitor of metalloproteinase-1 (TIMP-1)), oncoproteins (e.g., HER-2/neu, ras p21), growth
factor receptors (e.g., epidermal growth factor receptor (EGFR), platelet derived growth
factor receptor alpha (PDGFR-a)), metastasis proteins (e.g., urokinase-type plasminogen
activator (uPA)), tumor markers (e.g., carcinoembryonic antigen (CEA)), and tumor

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suppressors (e.g., p53). These methods may then be used, for example, as
diagnostic/prognostic tools, therapy selection for patients with a disease, monitoring the
status of a disease in a patient, and monitoring how a patient with a disease is responding to
a VEGF pathway-directed or other therapy. It would be advantageous to test patients (e.g.,
5 cancer patients) for serial changes in both total VEGF and additional proteins, such as
proteins that activate the VEGF pathway, as a means to enlarge the clinical perspective,
therapeutic resources, and diagnostic/prognostic parameters in order to select the optimal
therapeutic combinations for the most promising treatment outcomes.

[017] In another embodiment, the invention provides a test kit for monitoring the efficacy of
10 a therapeutic in a patient sample, comprising an antibody specific for a protein. In certain
embodiments, the kit further includes instructions for using the kit. In certain embodiments,
the kit may further include solutions for suspending or fixing the cells, detectable tags or
labels, solutions for rendering a polypeptide susceptible to the binding of an antibody,
solutions for lysing cells, or solutions for the purification of polypeptides. In a still further
15 embodiment, the antibody is specific for VEGF.

DESCRIPTION OF THE FIGURES

[018] Figure 1 illustrates the mean VEGF levels in patient populations at baseline
(pretreatment) and during treatment.

DETAILED DESCRIPTION OF THE INVENTION

20 [019] It is to be understood that this invention is not limited to the particular methodology,
protocols, cell lines, animal species or genera, constructs, and reagents described and as
such may vary. It is also to be understood that the terminology used herein is for the
purpose of describing particular embodiments only, and is not intended to limit the scope of
the present invention which will be limited only by the appended claims.

25 [020] It must be noted that as used herein and in the appended claims, the singular forms
"a," "and," and "the" include plural reference unless the context clearly dictates otherwise.
Thus, for example, reference to "a gene" is a reference to one or more genes and includes
equivalents thereof known to those skilled in the art, and so forth.

[021] Unless defined otherwise, all technical and scientific terms used herein have the
30 same meaning as commonly understood to one of ordinary skill in the art to which this

invention belongs. Although any methods, devices, and materials similar or equivalent to

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those described herein can be used in the practice or testing of the invention, the preferred
methods, devices and materials are now described.

[022] All publications and patents mentioned herein are hereby incorporated herein by
reference for the purpose of describing and disclosing, for example, the constructs and
5 methodologies that are described in the publications which might be used in connection with
the presently described invention. The publications discussed above and throughout the text
are provided solely for their disclosure prior to the filing date of the present application.
Nothing herein is to be construed as an admission that the inventors are not entitled to
antedate such disclosure by virtue of prior invention.

1 0 Definitions

[023] For convenience, the meaning of certain terms and phrases employed in the
specification, examples, and appended claims are provided below.

[024] The term "patient sample," as used herein, refers to a sample obtained from a
patient. The sample may be of any biological tissue or fluid. The sample may be a sample

15 which is derived from a patient. Such samples include, but are not limited to, blood, serum,
plasma, urine, saliva, semen, breast exudate, cerebrospinal fluid, tears, sputum, mucous,
lymph, cytosols, ascites, pleural effusions, peritoneal fluid, amniotic fluid, bladder washes,
and bronchioalveolar lavages, blood cells (e.g., white cells), tissue or biopsy samples (e.g.,
tumor biopsy), or cells therefrom. Biological samples may also include sections of tissues

20 such as frozen sections taken for histological purposes.

[025] The term "biomarker" encompasses a broad range of intra- and extra-cellular events
as well as whole-organism physiological changes. Biomarkers may be represent essentially
any aspect of cell function, for example, but not limited to, levels or rate of production of
signaling molecules, transcription factors, metabolites, gene transcripts as well as post-
25 translational modifications of proteins. Biomarkers may include whole genome analysis of
transcript levels or whole proteome analysis of protein levels and/or modifications.

[026] A biomarker may also refer to a gene or gene product which is up- or down-regulated
in a compound-treated, diseased cell of a subject having the disease compared to an
untreated diseased cell. That is, the gene or gene product is sufficiently specific to the
30 treated cell that it may be used, optionally with other genes or gene products, to identify,

predict, or detect efficacy of a small molecule. Thus, a biomarker is a gene or gene product
that is characteristic of efficacy of a compound in a diseased cell or the response of that
diseased cell to treatment by the compound.

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[027] The term "cancer" includes, but is not limited to, solid tumors, such as cancers of the
breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver,
skin, head and neck, thyroid, parathyroid, and their distant metastases. The term also
includes lymphomas, sarcomas, and leukemias.

5 [028] Examples of breast cancer include, but are not limited to, invasive ductal carcinoma,
invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.

[029] Examples of cancers of the respiratory tract include, but are not limited to, small-cell
and non-smal!-cell lung carcinoma, as well as bronchial adenoma and pleuropulmonary
blastema.

10 [030] Examples of brain cancers include, but are not limited to, brain stem and

hypophtalmic glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma,
as well as neuroectodermal and pineal tumor.

[031] Tumors of the male reproductive organs include, but are not limited to, prostate and
testicular cancer. Tumors of the female reproductive organs include, but are not limited to,
15 endometrial, cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma of the uterus.

[032] Tumors of the digestive tract include, but are not limited to, anal, colon, colorectal,
esophageal, gallbladder, gastric, pancreatic, rectal, small-intestine, and salivary gland
cancers.

[033] Tumors of the urinary tract include, but are not limited to, bladder, penile, kidney,
20 renal pelvis, ureter, and urethral cancers.

[034] Eye cancers include, but are not limited to, intraocular melanoma and
retinoblastoma.

[035] Examples of liver cancers include, but are not limited to, hepatocellular carcinoma
(liver cell carcinomas with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic
25 bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma.

[036] Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's
sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.

[037] Head-and-neck cancers include, but are not limited to, laryngeal / hypopharyngeal /
nasopharyngeal / oropharyngeal cancer, and lip and oral cavity cancer.

30 [038] Lymphomas include, but are not limited to, AIDS-related lymphoma, non-Hodgkin's
lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and lymphoma of the central
nervous system.

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[039] Sarcomas include, but are not linnited to, sarcoma of the soft tissue, osteosarcoma,
malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.

[040] Leukemias include, but are not limited to, acute myeloid leukemia, acute
lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and
5 hairy cell leukemia.

[041] The term "patient" or "subject" as used herein includes mammals (e.g., humans and
animals).

[042] The present invention is directed to quantitative immunoassays that measure the
levels of VEGF protein in patient samples. These assays may be useful for the selection of
10 a therapy for a patient with a disease associated with an activated VEGF pathway. As used
herein, an "activated VEGF pathway" is defined as a VEGF pathway activated by either
overexpression or mutation of VEGF protein and as such, encompasses upregulated and/or
mutationally stimulated VEGF pathways.

[043] Examples of neoplastic diseases associated with an activated VEGF pathway, as
15 well as precancers leading to neoplastic diseases, are the following: metastatic

medulloblastoma, gastrointestinal stromal tumors (GIST), dermatofibrosarcoma protruberans
(DFSP), chronic myeloproliferative diseases (CMPD), colorectal cancer, colon cancer, lung
cancer, non-small-cell lung cancer, small-cell lung cancer, acute myelogenous leukemia,
thyroid cancer, pancreatic cancer, bladder cancer, kidney cancer, melanoma, breast cancer,
20 prostate cancer, ovarian cancer, cervical cancer, head-and-neck cancer, brain tumors,

hepatocellular carcinoma, and hematologic malignancies. Thus, the levels of VEGF protein,
alone or in combination with levels of other proteins (e.g., other oncoproteins) may be used
to predict clinical outcome and/or as an aid in therapy selection.

[044] Thus, the present invention discloses and claims the application of an immunoassay
25 to quantitatively measure VEGF levels in patient samples (e.g., circulating VEGF levels) in
order to assess the likelihood that a patient suffering from cancer would benefit from
treatment with a multi-kinase inhibitor (e.g., Sorafenib).

[045] In one embodiment of the invention, VEGF protein is quantitated in patient samples
drawn at the time of diagnosis (e.g., renal cell carcinoma), as well as subsequent time points
30 post-treatment (e.g., day 31 of the first cycle of treatment, day 1 of the third cycle of

treatment). Such patient samples may be, for example, blood, serum, plasma, urine, saliva,
semen, breast exudate, cerebrospinal fluid, tears, sputum, mucous, lymph, cytosols, ascites,
pleural effusions, amniotic fluid, bladder washes, and bronchioalveolar lavages, among other

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body fluid samples. The patient samples be fresti or frozen, and may be treated with
heparin, citrate, or EDTA.

[046] As an example of an immunoassay that may be used in the methods of the invention
is a sandwich ELISA. However, it can be appreciated that other methods, in addition to
5 those disclosed herein, may be used to quantify VEGF protein in patient samples.

Furthermore, a number of detection methods may be used to visualize the VEGF protein,
such as luminescent labels.

[047] Many formats may be adapted for use with the methods of the present invention. For
example, the detection and quantitation of VEGF protein in patient samples may be

10 performed, by enzyme-linked immunosorbent assays, radioimmunoassays, dual antibody
sandwich assays, agglutination assays, fluorescent immunoassays, immunoelectron and
scanning microscopy, among other assays commonly known in the art. The quantitation of
VEGF protein in such assays may be adapted by conventional methods known in the art. In
one embodiment, serial changes in circulating VEGF protein levels may be detected and

15 quantified by a sandwich assay in which the capture antibody has been immobilized using
conventional techniques on the surface of the support.

[048] Suitable supports include, for example, synthetic polymer supports, such as
polypropylene, polystyrene, substituted polystyrene, polyacrylamides (such as polyamides
and polyvinylchloride), glass beads, agarose, and nitrocellulose.

20 [049] An example of an ELISA sandwich immunoassay that may be used in the methods of
the present invention, uses purified mouse anti-human VEGF monoclonal antibody as the
capture antibody and biotinylated goat anti-human VEGF polyclonal antibody as the detector
antibody. The capture monoclonal antibody is immobilized on microtiter plate wells. Diluted
human serum/plasma samples or VEGF standards (recombinant wild-type VEGF protein)

25 are incubated in the wells to allow binding of VEGF antigen by the capture monoclonal

antibody. After washing of wells, the immobilized VEGF antigen is exposed to a biotinylated
detector antibody after which the wells are again washed. A streptavidin-horseradish
peroxidase conjugate is then added. After a final wash, TMB Blue Substrate is added to the
wells to detect bound peroxidase activity. The reaction is stopped by the addition of 2.5 N

30 sulfuric acid, and the absorbance is measured at 450 nm. Correlating the absorbance

values of samples with the VEGF standards allows the determination of a quantitative value
of VEGF in pg/ml of serum or plasma.

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[050] It can be appreciated that other proteins (e.g., inhibitors, oncoproteins, growth factor
receptors, angiogenic factors, metastasis proteins, tunnor markers, tumor suppressors,
proteins associated with the VEGF pathway) may be suitable for detection and quantitation
in combination with VEGF. For example, other proteins suitable for testing along with VEGF
5 include tissue inhibitor of metalloproteinase-1 (TIMP-1), HER-2/neu, ras p21, epidermal
growth factor receptor (EGFR), platelet derived growth factor receptor alpha, vascular
endothelial growth factor (VEGF), urokinase-type plasminogen activator (uPA),
carcinoembryonic antigen (CEA), and p53. These other proteins may be detected using
assays that are known to one of skill in the art. For example, immunoassays for the
10 quantitation of HER-2/neu and TlMP-1 are commercially available, such as the Oncogene
Science TlMP-1 ELISA (Oncogene Science, Cambridge, MA (USA)) which can detect ng/ml
values of TIMP-1 levels in human serum or plasma.

[051] Monitoring the pretreatment levels of VEGF may be indicative of clinical outcome
following treatment with a multi-kinase inhibitor (e.g., Sorafenib). One method of evaluating
15 a clinical outcome may be assessment of response rate (RR), complete response (OR),
partial response (PR), stable disease (SD), clinical benefit (including complete response
(OR), partial response (PR), and stable disease (SD)), time to progression (TTP),
progression free survival (PFS), and overall survival (OS).

[052] The term "antibody" herein is used in the broadest sense and specifically covers
20 monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies,
multispecific antibodies (e.g., bispecific antibodies), and antibody fragments. Antibodies
useful according to the methods of the invention may be prepared by conventional
methodology and/or by genetic engineering. For example, antibodies according to the
invention include those antibodies that bind to VEGF.

25 [053] "Antibody fragments" comprise a portion of a full length antibody, generally the

antigen binding or variable domain thereof. Examples of antibody fragments include Fab,
Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody
molecules; biospecific antibodies; and multispecific antibodies formed from antibody
fragments.

30 [054] The term "monoclonal antibody" as used herein refers to an antibody obtained from a
population of substantially homogeneous antibodies, that is, individual antibodies comprising
an identical population except for possible naturally occurring mutations that may be present
in minor amounts. Monoclonal antibodies are highly specific, that is, directed against a

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single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody
preparations which typically Include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed against a single determinant
on the antigen. The modifier "monoclonal" indicates the character of the antibody as being
5 obtained from a substantially homogeneous population of antibodies, and is not to be

construed as requiring production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present invention may be made
by the hybridoma method first described by Kohler, et al., (Nature 256:495, 1975), or may be
made by recombinant DNA methods {see, e.g., U.S. Patent No. 4,816,567). Monoclonal
10 antibodies may also be isolated from phage antibody libraries using the techniques

described in, for example, Clackson, et al., (Nature 352:624-628,1991) and Marks, et al., (J.
Mol. Biol. 222:581-597, 1991).

[055] The monoclonal antibodies herein also include "chimeric" antibodies
(Immunoglobulins) in which a portion of the heavy and/or light chain is identical with or

15 homologous to corresponding sequences in antibodies derived from a particular species or
belonging to a particular antibody class or subclass, while the remainder of the chain(s) is
identical with or homologous to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Patent No.

20 4,816,567; and Morrison, et al., Proc. Natl. Acad. Sci. USA 81:6851-6855, 1984).

[056] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies
which contain minimal sequence derived from non-human Immunoglobulin. For the most

■ part, humanized antibodies are human immunoglobulins (recipient antibody) In which

hypervariable region residues of the recipient are replaced by hypervarlable region residues

25 from a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman

primate having the desired specificity, affinity, and capacity. In some instances, framework
region (FR) residues of the human immunoglobulin may be replaced by corresponding non-
human residues. Furthermore, humanized antibodies may comprise residues which are not
found In the recipient antibody or in the donor antibody. Such modifications are made to

30 further refine antibody performance. In general, the humanized antibody may comprise

substantially all of at least one or typically two variable domains, in which all or substantially
all of the hypervariable regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FRs are those of a human immunoglobulin sequence. The
humanized antibody optionally also may comprise at least a portion of an immunoglobulin

35 constant region (Fc), typically that of a human immunoglobulin. For a review, see Jones, et

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al., (Nature 321:522-525, 1986); Reichmann, et al., (Nature 332:323-329, 1988); and Presta,
(Curr. Op. Struct. Biol. 2:593-596, 1992).

[057] "Single-chain Fv" or "sFv" antibody fragments connprise the Vh and Vl domains of
antibody, wherein these domains are present in a single polypeptide chain. Generally, the
5 Fv polypeptide further comprises a polypeptide linker between the Vh and Vl domains which
enables the sFv to form the desired structure for antigen binding. For a review, see
Pluckthun (The Pharmacoloqv of Monoclonal Antibodies , Vol. 113, Rosenburg and Moore
eds. Springer-Verlag, New York, pp. 269-315, 1994).

[058] The term "diabodies" refers to small antibody fragments with two antigen-binding
10 sites, which fragments comprise a heavy chain variable domain (Vh) connected to a light
chain variable domain (Vl) in the same polypeptide chain (Vh-Vl). By using a linker that is
too short to allow pairing between the two domains on the same chain, the domains are
forced to pair with the complementary domains of another chain and create two antigen-
binding sites. Diabodies are described more fully in, for example, EP 404,097; WO
15 93/11161; and Hollinger, et al., (Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993).

[059] The expression "linear antibodies" refers to the antibodies described in Zapata, et al.,
(Protein Eng. 8(1 0):1 057-1 062, 1995). Briefly, such antibodies comprise a pair of tandem Fd
segments (Vh-Ch1-Vh-Ch1) which form a pair of antigen binding regions. Linear antibodies
can be bispecific or monospecific.

20 [060] Representative monoclonal antibodies useful according to this invention include

mouse anti-human total VEGF monoclonal antibodies, such as those found in the Oncogene
Science sandwich ELISA kit designed to measure human VEGF. Monoclonal antibodies
useful according to this invention serve to identify VEGF proteins in various laboratory
prognostic tests, for example, in clinical samples.

25 [061] General texts describing additional molecular biological techniques useful herein.
Including the preparation of antibodies include Berger and Kimmel ( Guide to Molecular
Cloning Techniques, Methods in Enzvmoloqy . Vol. 152, Academic Press, Inc.); Sambrook, et
al., ( Molecular Cloning: A Laboratory Manual , (Second Edition, Cold Spring Harbor
Laboratory Press; Cold Spring Harbor, N.Y.; 1989) Vol. 1-3); Current Protocols in Molecular

30 Biology , (F. M. Ausabel et al. [Eds.], Current Protocols, a joint venture between Green
Publishing Associates, Inc. and John Wiley & Sons, Inc. (supplemented through 2000));
Harlow et al., ( Monoclonal Antibodies: A Laboratory Manual . Cold Spring Harbor Laboratory
Press (1988), Paul [Ed.]); Fundamental Immunology , (Lippincott Williams &Wilkins (1998));

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and Harlow, et aL, ( Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory
Press (1998)).

[062] The antibodies useful according to this invention to identify VEGF proteins may be
labeled in any conventional manner. An example of a label is horseradish peroxidase, and
an example of a method of labeling antibodies is by using biotin-strepavidin complexes.

[063] As appropriate, antibodies used in the immunoassays of this invention that are used
as tracers may be labeled in any manner, directly or indirectly, that results in a signal that is
visible or can be rendered visible. Detectable marker substances include radionuclides,
such as ^H, ''^^l, and ^^^1; fluorescers, such as, fluorescein isothiocyanate and other
fluorochromes, phycobiliproteins, phycoerythin, rare earth chelates, Texas red, dansyl and
rhodamine; colorimetric reagents (chromogens); electron-opaque materials, such as colloidal
gold; bioluminescers; chemiluminescers; dyes; enzymes, such as, horseradish peroxidase,
alkaline phosphatases, glucose oxidase, glucose-6-phosphate dehydrogenase,
acetylcholinesterase, alpha beta-galactosidase, among others; coenzymes; enzyme
substrates; enzyme cofactors; enzyme inhibitors; enzyme subunits; metal ions; free radicals;
or any other immunologically active or inert substance which provides a means of detecting
or measuring the presence or amount of immunocomplex formed. Exemplary of enzyme
substrate combinations are horseradish peroxidase and tetramethyl benzidine (TMB), and
alkaline phosphatases and paranitrophenyl phosphate (pNPP).

[064] Another detection and quantitation systems according to this invention produce
luminescent signals, bioluminescent (BL) or chemiluminescent (CL). In chemiluminescent
(CL) or bioluminescent (BL) assays, the intensity or the total light emission is measured and
related to the concentration of the unknown analyte. Light can be measured quantitatively
using a luminometer (photomultiplier tube as the detector) or charge-coupled device, or
qualitatively by means of photographic or X-ray film. The main advantages of using such
assays is their simplicity and analytical sensitivity, enabling the detection and/or quantitation
of very small amounts of analyte.

[065] Exemplary luminescent labels are acridinium esters, acridinium sulfonyl
carboxamides, luminol, umbeiiiferone, isoluminol derivatives, photoproteins, such as
aequorin, and luciferases from fireflies, marine bacteria, Varaulia and Renilla. Luminol can
be used optionally with an enhancer molecule such as 4-iodophenol or 4-hydroxy-cinnamic
acid. Typically, a CL signal is generated by treatment with an oxidant under basic
conditions.

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[066] Additional luminescent detection systems are tliose wlierein tine signal (detectable

marker) is produced by an enzymatic reaction upon a substrate. CL and BL detection

schemes have been developed for assaying alkaline phosphatases (AP), glucose oxidase,

glucose 6-phosphate dehydrogenase, horseradish peroxidase (HRP), and xanthine-oxidase

labels, among others. AP and HRP are two enzyme labels which can be quantitated by a

range of CL and BL reactions. For example, AP can be used with a substrate, such as an

adamantyl 1,2-dioxetane aryl phosphate substrate (e.g. AMPPD or CSPD; Kricka, L.J.,

"Chemiluminescence and Bloluminescence, Analysis by," Molecular Biology and

Biotechnoloav: A Co mprehensive Desk Reference (ed. R.A. Meyers) (VCH Publishers;

N.Y., N.Y.; 1995)); for example, a disodium salt of 4-methoxy-4-(3-phosphatephenyl) spiro

[1,2-dioxetane-3,2'-adamantane], with or without an enhancer molecule such as

1-(trioctylphosphonium methyl)-4- (tributylphosphonium methyl) benzene diochloride. HRP

is may be used with substrates, such as, 2',3',6 -trifluorophenyl-methoxy-IO-methylacridan-
Q-carboxylate.

[067] CL and BL reactions may be adapted for analysis not only of enzymes, but also of
other substrates, cofactors, inhibitors, metal ions, and the like. For example, luminol, firefly
luciferase, and marine bacterial luciferase reactions are indicator reactions for the production
or consumption of peroxide, ATP, and NADPH, respectively. They may be coupled to other
reactions involving oxidases, kinases, and dehydrogenases, and may be used to measure
any component of the coupled reaction (enzyme, substrate, cofactor).

1068] The detectable marker may be directly or indirectly linked to an antibody used in an
assay of this invention. Exemplary of an indirect linkage of the detectable label is the use of
a binding pair between an antibody and a marker or the use of a signal amplification system.

[069] Examples of binding pairs that may be used to link antibodies to detectable markers
are biotin/avidin, streptavidin, or anti-biotin; avidin/anti-avidin; thyroxine/thyroxine-binding
globulin; antigen/antibody; antibody/ anti-antibody; carbohydrate/lectins; hapten/anti-hapten
antibody; dyes and hydrophobic molecules/hydrophobic protein binding sites; enzyme
inhibitor, coenzyme or cofactor/enzyme; polynucleic acid/homologous polynucleic acid
sequence; fluorescein/anti- fluorescein; dinitrophenol/anti-dinitrophenol; vitamin B12/intrinsic
factor; cortisone, cortisol/cortisol binding protein; and ligands for specific receptor
protein/membrane associated specific receptor proteins.

[070] Various means for linking labels directly or indirectly to antibodies are known in the
art. For example, labels may be bound either covalently or non-covalently. Exemplary

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antibody conjugation methods are described in Avarmeas, et al., Scan. J. Immunol. 8(Suppl.
7): 7, 1978); Bayer, etal., Metli. Enzymol. 62:308, 1979; Chandler, et al., J. Immunol. Meth.
53:187, 1982; Ekeke and Abuknesha, J. Steroid Biochem. 11:1579, 1979; Engvall and
Perlmann, J. Immunol. 109:129, 1972; Geoghegan, et al., Immunol, Comm. 7:1, 1978; and
5 Wilson and Nakane, Immunofluorescence and Related Techniques , Elsevier/North Holland
Biomedical Press; Amsterdam (1978).

[071] Depending upon the nature of the label, various techniques may be employed for
detecting and quantitating the label. For fluorescers, a large number of fluorometers are
available. For chemiluminescers, luminometers or films are available. With enzymes, a
10 fluorescent, chemiluminescent, or colored product may be determined or measured
fluorometrically, luminometrically, spectrophotometrically, or visually.

[072] Various types of chemiluminescent compounds having an acridinium,
benzacridinium, or acridan type of heterocyclic ring systems are other examples of labels.
Examples of acridinium esters include those compounds having heterocyclic rings or ring
15 systems that contain the heteroatom in a positive oxidation state including such ring systems
as acridinium, benz[a]acridinium, benz[b]acridinium, benz[c]acridinium, a benzimidazole
cation, quinolinium, isoquinolinium, quinolizinium, a cyclic substituted quinolinium,
phenanthridinium, and quinoxalinium.

[073] The tracer may be prepared by attaching to the selected antibody either directly or
20 indirectly a reactive functional group present on the acridinium or benzacridinium ester, as is
well known to those skilled in the art (see, e.g., Weeks, et al., Clin. Chem. 29(8): 1474-1 479,
1983). Examples of compounds are acridinium and benzacridinium esters with an aryl ring
leaving group and the reactive functional group present in either the para or the meta
position of the aryl ring, (see, e.g., U.S. Patent No. 4,745,181 and WO 94/21823).

25 [074] As used herein, "VEGF pathway-directed therapies" include any therapies that are
targeted to the VEGF pathway, including inhibition of VEGF protein expression (e.g.,
antisense oligonucleotides), prevention of membrane localization essential for VEGFR
activation, or inhibition of downstream effectors of VEGFR (e.g., Raf serine/threonine
kinases). VEGF pathway-directed therapies include multi-kinase inhibitors, tyrosine kinase

30 inhibitors, monoclonal antibodies, and bis-aryl ureas.

[075] An example of a kinase inhibitor is the bis-aryl urea Sorafenib, a small molecule and
novel dual-action inhibitor of both Raf (a protein-serine/threonine kinase) and VEGFR
(vascular endothelial growth factor receptor, a receptor tyrosine kinase), and consequently

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an inhibitor of both tumor cell proliferation and angiogenesis (Onyx Pharmaceuticals,
Richmond, CA, and Bayer Pharmaceuticals Corporation, West Haven, CT (USA); Lyons, et
al., Endocrine-Related Cancer 8:21i9-225, 2001). In addition, Sorafenib has been found to
inhibit several other receptor tyrosine kinases involved in tumor progression and
5 neovascularization, including PDGFR-)ff, Flt-3, and c-KIT. PD1 66285 (Pfizer, Groton, CT), a
general tyrosine kinase inhibitor, can antagonize both PDGF and FGF-2-mediated
responses (Bansai, et al., J. Neuroscience Res. 74(4):486-493, 2003).

[076] Other exemplary therapies that target the VEGF pathway include: Sutent/SU1 1248,
PTK 787, MLN518, PKC-412, CDP860, and XL9999. Sutent/SU11248 (sunitinib malate; an

10 indoline-2-one) (Pfizer, Groton, CT) targets receptor tyrosine kinases (RTKs) including
PDGFR, with anti-angiogenic and anti-tumor effects. PDGFR plays a significant role in
fostering angiogenesis by regulating the proliferation and migration of pericytes, cells that

- support blood vessels, and Sutent/SU11248 is believed to inhibit PDGFR's angiogenic
action.

15 [077] PTK 787 (Novartis, Basel, Switzerland and Schering AG, Berlin, Germany) is a oral
small molecule antl-angiogenesis agent (anilinophthalazine) active against PDGFR, as well
as against VEGFR and c-Kit tyrosine kinase receptors (see, e.g., Garcia-Echevera and
Fabbro, Mini Reviews in Medicinal Chemistry 4(3):273-283, 2004).

[078] MLN518 (formerly known as CT53518; Millenium Pharmaceuticals, Cambridge, MA)
20 is an oral, small molecule designed to inhibit type III receptor tyrosine kinases (RTKs),
including PDGFR, FLT3, and c-Kit.

[079] PKC-412 [midostaurin; N-benzoyl-staurosporine (a derivative of staurosporine, a
product of Streptomyces bacteria); Novartis, Basel, Switzerland) inhibits PDGFR, VEGFR
and multiple protein kinase Cs, "which makes it especially attractive in patients with wild-type
25 KIT with mutations in PDGFR" (PKC 412-An Interview with Charles Blanke, MD, FACP

(www.gistsupport.org/pkc412.html); see also Reichardt, et al., J. Clin. Oncol. 23(16S):3016,
2005).

[080] XL999 (one of several Spectrum Selective Kinase Inhibitors™ (SSKIs) from Exelixis
(South San Francisco, CA, USA)] inhibits VEGFR, as well as other RTKs, such as PDGFR-
30 beta, FGFR1, and FLT3.

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EXAMPLES

[081] The structures, materials, compositions, and methods described herein are intended
to be representative examples of the invention, and it will be understood that the scope of
5 the invention is not limited by the scope of the examples. Those skilled in the art will

recognize that the invention may be practiced with variations on the disclosed structures,
materials, compositions and methods, and such variations are regarded as within the ambit
of the invention.

Example 1. Solid Phase Sandwich Microtiter ELISA for Human Serum and Plasma
1 0 Sample Preparation

[082] Suitable samples for analysis by the VEGF ELISA include human plasma treated
with heparin, citrate, or EDTA, and human serum. Due to possible interfering factors;
special care must be taken in the preparation and assay of human serum and plasma. Any
flocculant material should be removed from samples by microcentrifugation prior to dilution.
15 The initial concentration of the serum or plasma specimen to be examined should be about
1 2-1 3% (a 1 :8 dilution of specimen in sample diluent). For example, 40 |al of sample may be
diluted into 280 ^il of sample diluent, and 100 pi added to the microplate wells.

Assay Procedure

20 [083] The following ELISA protocol is that used for the sandwich ELISA (Oncogene
Science, Cambridge, MA) to measure human VEGF in human plasma or serum.

1 . Prepare a working solution (1X) of Platewash (Provided as part of the assay kit).

2. Add prediluted samples and Controls, and each of the six VEGF Standards (0 to 8000
pg/mL) in duplicate by pipetting 100 pL into the appropriate wells using clean pipet tips

25 for each sample and Standard. Add Standard 0 to one additional well to be used for

determination of Substrate blank.

3. Cover wells with clean plastic wrap or plate sealer. Incubate microtiter plate for 1 .5
hours at 37°C.

4. Carefully remove the plastic wrap or plate sealer. Wash wells using 300 pL per well
30 with six cycles of Platewash buffer (Wash for three cycles, rotate the plate 180°, and

wash for three more cycles).

5. Pipet 100 pL of the Detector Antibody into all wells except the Substrate blank well,
which is left empty. Cover the wells with a fresh piece of plastic wrap. Incubate
microtiter plate for 1 hour at 37°C.

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6. Prepare Working Conjugate by diluting an appropriate volume of Conjugate
Concentrate (1:50 dilution) into Conjugate Diluent.

7. Wash wells as in Step 4. Proceed immediately to Step 8.

8. Pipet 100 pL of Working Conjugate into all wells except the Substrate blank well, which
is left empty. Cover the wells with a fresh piece of plastic wrap. Incubate the microtiter
plate at room temperature (20-27°C) for 1 hour.

9. Prepare Working Substrate by combining equal parts of Solution A and Solution B.

Six mL of each Substrate solution will provide 12 mL of Working Substrate, sufficient to
develop one microtiter plate. Adjust volume of Working Substrate based on number of
strips used. Mix well.

10. Dispense Working Substrate into a clean reagent troiigh and allow it to come to room
temperature.

1 1 . Wash wells as in Step 5. CAUTION: Do not allow plates to dry out. Proceed
immediately to Step 12.

12. Pipet 100 pL of Working Substrate into all wells and cover the plate with plastic wrap or
plate sealer. Incubate the microtiter plate at room temperature (20-27°C) for

45 minutes.

1 3. Pipet 1 00 pL of Stop Solution into all wells.

14. Measure absorbance in each well using a spectrophotometric plate reader at a
wavelength of 650 nm. Wells should be read within 30 minutes of adding the Stop
Solution.

Standard Curves

[084] Quantitative analyses were made by constructing a standard curve using VEGF
standard (recombinant human VEGF) at 6 different concentrations of 0, 150, 1000. 3000,
5000, and 8000 pg/ml.

Human Serum and Plasma Samples

[085] Frozen plasma samples were obtained from patients with confirmed renal cell
carcinoma prior to treatment with Sorafenib.

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Example 2. Plasma from Renal Cell Carcinoma Patients

[086] Duplicate samples were used to measure the VEGF level using a VEGF ELISA (R&D
Systems, Minneapolis, MN) per the manufacturers directions. The mean value of the
duplicate measurements was determined for each patient. The mean levels of VEGF are
5 reported in Table 1 for three time points, Baseline (pretreatment). Cycle 1 Day 21, and Cycle
3 Day 1 for both a group of patients treated with Sorafenib and a group of patients treated
with a placebo. The sarne data is shown in Figure 1. The results shown that the Sorafenib-
treated patient group have VEGF levels that increase significantly from baseline (p « 0.01
using a paired t-test) at both time points, but this does not occur in the placebo-treated group
10 (p > 0.05).

Table 1: VEGF

Median VEGF (pg/ml)
(Number of patients)

Baseline

Cycle 1
Day 21

Cycle 3
Day 1

237.4

301.6

309.0

Sorafenib

(149)

(196)

(197)

174.1

186.2

182.5

Placebo

(102)

(128)

(132)

15

[087] The description of the foregoing embodiment of the invention has been presented for
purposes of illustration and description. It is not intended to be exhaustive or to limit the
invention to the precise form disclosed, and obviously many modifications and variations are
possible in light of the above teachings. The embodiments were chosen and described in
20 order to explain the principles of the invention and its practical application to enable thereby
others skilled in the art to utilize the invention in various embodiments and with various
modifications as are suited to the particular use contemplated. All references cited herein
are hereby incorporated by reference.

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Claims

1 . A method for monitoring the status of a disease associated with the VEGF pathway in
a patient, and/or monitoring how a patient with said disease is responding to a therapy
comprising Immunologically detecting and quantifying serial changes in VEGF protein
levels in patient samples taken overtime, wherein increasing levels of VEGF protein
overtime indicate disease progression or a negative response to said therapy, and
wherein decreasing levels of VEGF protein overtime indicate disease remission or a
positive response to said therapy.

2. The method of claim 1 , wherein said therapy is selected from multi-kinase inhibitors,
tyrosine kinase inhibitors, monoclonal antibodies, and bis-aryl ureas.

3. The method of claim 1 , wherein said therapy is a VEGF pathway-directed therapy.

4. The method of claim 3, wherein said VEGF pathway-directed therapy is the tyrosine
kinase inhibitor imatinib mesylate or the bis-aryl urea Sorafenib.

5. The method of claim 1, wherein said disease is a preneoplastic/neoplastic disease.

6. The method of claim 5, wherein said preneoplastic/neoplastic disease is selected from
the group consisting of metastatic medulloblastoma, dermatoflbrosarcoma
protruberans, gastrointestinal stromal tumors, colorectal cancer, colon cancer, lung
cancer, non-small-cell lung cancer, small-cell lung cancer, chronic myeloproliferative
diseases, acute myelogenous leukemia, thyroid cancer, pancreatic cancer, bladder
cancer, kidney cancer, melanoma, breast cancer, prostate cancer, ovarian cancer,
cervical cancer, head-and-neck cancer, brain tumors, hepatocellular carcinoma,
hematologic malignancies, and precancers leading to the aforementioned cancers.

7. The method of claim 1 which is further prognostic for said disease, wherein said levels
of VEGF protein in the patienf s samples are indicative of a better or poorer prognosis
for said patient.

8. The method of claim 7, wherein said prognosis Is a clinical outcome selected from the
group consisting of response rate (RR), complete response (CR), partial response
(PR), stable disease (SD), time to progression (TTP), progression free survival (PFS),
overall survival (OS), and clinical benefit, which comprises complete response (CR),
partial response (PR), and stable disease (SD).

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9. The method of claim 7, wherein increasing levels of VEGF are indicative of a greater
probability of early recurrence or metastasis.

10. The method of claim 1, wherein said patient's samples are pretreatment samples.

1 1 . The method of claim 1 , wherein said patient sample is selected from the group
consisting of blood, serum, plasma, urine, saliva, semen, breast exudate, cerebrospinal
fluid, tears, sputum, mucous, lymph, cytosols, ascites, pleural effusions, amniotic fluid,
bladder washes and bronchioalveolar lavages.

1 2. The method of claim 1 , wherein said patient sample is serum or plasma.

13. The method of claim 1, wherein said immunological detection and quantitation is by an
immunoassay in the form of a sandwich ELISA or equivalent assay.

14. The method of claim 13, wherein the sandwich ELISA or equivalent assay comprises
the use of one or more monoclonal antibodies that selectively bind the VEGF protein.

1 5. The method of claim 1 , further comprising the use of an immunoassay to detect or
detect and quantify levels of one or more other proteins in the patient's samples.

16. The method of claim 15, wherein said other protein is or said other proteins are
selected from the group consisting of inhibitors, oncoproteins, growth factor receptors,
angiogenic factors, metastasis proteins, tumor markers, and tumor suppressors.

17. The method of claim 16 wherein said inhibitor is tissue inhibitor of metalloproteinase-1
(TIMP-1), said oncoproteins are selected from the group consisting of HER-2/neu and
ras p21, said growth factor receptors are selected from the group consisting of
epidermal growth factor receptor (EG PR) and platelet derived growth factor receptor
alpha (PDGFR-a), said angiogenic factor is vascular endothelial growth factor (VEGF),
said metastasis protein is urokinase-type plasminogen activator (uPA), said tumor
marker is carcinoembryonic antigen (CEA), and said tumor suppressor is p53.

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18. A method of therapy selection for a human patient with a disease, comprising:

(a) immunologically detecting and quantifying the average level of VEGF protein in
control samples taken from individuals of a control population;

(b) immunologically detecting and quantifying serial changes in VEGF protein
levels in equivalent patient samples taken from the patient over time;

(c) comparing the levels of VEGF protein in the patient's samples to the average
level of VEGF protein in the control samples; and

(d) determining whether to use conventional therapy and/or VEGF pathway-
directed therapy to treat the patient based upon the differences between the
levels of VEGF protein in the patient's samples and the average level of VEGF
protein in the control samples, and in view of the serial changes among the
levels of VEGF protein in the patient's samples.

19. The method of claim 18, wherein said patient's samples are pretreatment samples.

20. The method of claim 18 which is further prognostic for said disease, wherein said
levels of VEGF protein in the patient's samples are indicative of a better or poorer
prognosis for said patient.

21 . The method of claim 20, wherein said prognosis is a clinical outcome selected from the
group consisting of response rate (RR), complete response (CR), partial response
(PR), stable disease (SD), time to progression (TTP), progression free survival (PFS),
overall survival (OS), and clinical benefit, which comprises complete response (CR),
partial response (PR), and stable disease (SD).

22. The method of claim 18, wherein said disease is a preneoplastic/neoplastic disease.

23. The method of claim 22, which said preneoplastic/neoplastic disease is selected from
the group consisting of metastatic medulloblastoma, dermatofibrosarcoma
protruberans, gastrointestinal stromal tumors, colorectal cancer, colon cancer, lung
cancer, non-small-cell lung cancer, small-cell lung cancer, chronic myeloproliferative
diseases, acute myelogenous leukemia, thyroid cancer, pancreatic cancer, bladder
cancer, kidney cancer, melanoma, breast cancer, prostate cancer, ovarian cancer,
cervical cancer, head-and-neck cancer, brain tumors, hepatocellular carcinoma,
hematologic malignancies, and precancers leading to the aforementioned cancers.

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10

30

24. The method of claim 18, wherein the patient samples are from a cancer patient who
has not responded to treatment.

25. The method of claim 18, further comprising the use of an immunoassay to detect or
detect and quantify levels of one or more other proteins in the subject's samples.

26. The method of claim 25, wherein said other protein is or said other proteins are
selected from the group consisting of inhibitors, oncoproteins, growth factor receptors,
angiogenic factors, metastasis proteins, tumor markers, and tumor suppressors.

27. The method of claim 26 wherein said inhibitor is tissue inhibitor of metalloproteinase-1
(TIMP-1), said oncoproteins are selected from the group consisting of HER-2/neu and
ras p21, said growth factor receptors are selected from the group consisting of
epidermal growth factor receptor (EGFR) and platelet derived growth factor receptor

15 alpha (PDGFR-a), said angiogenic factor is vascular endothelial growth factor (VEGF),

said metastasis protein is urokinase-type plasminogen activator (uPA), said tumor
marker is carcinoembryonic antigen (CEA), and said tumor suppressor is p53.

28. A diagnostic method to detect a disease associated with a VEGF pathway in a patient
20 comprising:

(a) immunologically detecting and quantifying the average level of VEGF protein in
control samples taken from individuals of a control population;

(b) immunologically detecting and quantifying serial changes in VEGF protein in
samples of a patient sample taken from a patient over time; and

25 (c) comparing the levels of VEGF protein in the patient's samples to the average

level of VEGF protein in the control samples;

wherein a level of VEGF protein in the patient's samples that is above the average
level of VEGF protein in the control samples is indicative of an activated VEGF
pathway and the presence of disease in the patient.

29. The method of claim 28, wherein said immunological detection and quantification of
steps (a) and (b) is by an immunoassay in the form of a sandwich ELISA or equivalent
assay.

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30. The method of claim 28 which is further prognostic for said disease, wherein said
levels of VEGF protein in the patient's samples are indicative of a better or poorer
prognosis for said patient.

31 . The method of claim 30, wherein said prognosis is a clinical outcome selected from the
group consisting of response rate (RR), complete response (CR), partial response
(PR), stable disease (SD), time to progression (TTP), progression free survival (PFS),
overall survival (OS), and clinical benefit, which comprises complete response (CR),
partial response (PR), and stable disease (SD).

32. The method of claim 28, wherein said disease is a preneoplastic/neoplastic disease.

33. The method of claim 32, wherein said preneoplastic/neoplastic disease associated with
an activated PDGF pathway is selected from the group consisting of metastatic
medulloblastoma, gastrointestinal stromal tumors, dermatofibrosarcoma protruberans,
colorectal cancer, colon cancer, lung cancer, non-small-cell lung cancer, small-cell lung
cancer, chronic myeloproliferative diseases, acute myelogenous leukemia, thyroid
cancer, pancreatic cancer, bladder cancer, kidney cancer, melanoma, breast cancer,
prostate cancer, ovarian cancer, cervical cancer, head-and-neck cancer, brain tumors,
hepatocellular carcinoma, hematologic malignancies, and precancers leading to the
aforementioned cancers.

34. The method of claim 28, further comprising the use of an immunoassay to detect or
detect and quantify levels of one or more other proteins in the patient's samples.

35. The method of claim 34, wherein said other protein is or said other proteins are
selected from the group consisting of inhibitors, oncoproteins, growth factor receptors,
angiogenic factors, metastasis proteins, tumor markers, and tumor suppressors.

36. The method of claim 35 wherein said inhibitor is tissue inhibitor of metalloproteinase-1
(TIMP-1), said oncoproteins are selected from the group consisting of HER-2/neu and
ras p21 , said growth factor receptors are selected from the group consisting of
epidermal growth factor receptor (EGFR) and platelet derived growth factor receptor
alpha (PDGFR-a), said angiogenic factor is vascular endothelial growth factor (VEGF),
said metastasis protein is urokinase-type plasminogen activator (uPA), said tumor
marker is carcinoembryonic antigen (CEA), and said tumor suppressor is p53.

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1 / 1

CZU Sorafenib — M — Placebo
330

280 -

130 -

80 i — ' ' — I — ' ' — \ — '

Baseline Cycle 1, Cycle 3,

Day 21 Day 1

FIG. 1

SUBSTITUTE SHEET (RULE 26)